Hybrid vigor is a phenomenon by which the progeny of a cross between two inbred lines has a higher yield potential than either one of the parents. Hybrids can yield up to 10-30% more than the best non-hybrid varieties, and are a favored option for increasing yield.
The most widely used system for hybrid pepper production is the three line system: (a) a male sterile and female fertile line called the cytoplasmic male sterile (CMS) line because it carries a male sterility-conferring mutation in the cytoplasmic component of the genome; (b) a maintainer line; and (c) a restorer line. The maintainer and restorer lines are male fertile as well as female fertile. The CMS and maintainer lines are practically identical with respect to the nuclear component of the genome (and are often referred to as iso-nuclear lines) but differ from each other with respect to the cytoplasmic component of the genome. The male sterility of the CMS line is maternally inherited and is most likely due to a mutation in the mitochondrial DNA. The CMS line, being female fertile, can be propagated by fertilization with pollen emanating from the maintainer line. Since the cytoplasmic component of the genome is not transferred through pollen, the progeny of such a cross would inherit the cytoplasm only from the CMS line and would therefore be male sterile. The nuclear component of the genome of the progeny would also be identical to that of the CMS line, even though half of it is inherited from the maintainer line, as there is no difference between these two lines with respect to this component of the genome.
The hybrid seeds are produced in a cross of the CMS line with another inbred parental line, called the restorer line, which as indicated above is male fertile and female fertile. In this cross, the CMS line serves as the female parent while the restorer line is the male parent. The restorer line also carries Rf (restorer of fertility) gene/s in its nuclear genome which will restore male fertility to a plant whose cytoplasm has been inherited from the CMS line. The hybrid seeds therefore would be produced. The CMS and restorer lines are appropriately chosen such that the hybrids exhibit sufficient hybrid vigor (or heterosis) to produce substantially higher yields than inbred varieties.
The CMS in pepper (Capsicum annuum L.) was first documented by Peterson (Peterson P A (1958) “Cytoplasmically inherited male sterility in Capsicum.” Amer Nat., 92:111-119) for PI 164835, which was introduced from India. Since then, commercial seed companies have utilized this trait to produce hybrid F1, seeds in the field. Male sterile (S-) cytoplasm of Peterson's lines is the only common source of CMS used to produce hybrid F1 pepper seeds.
One example of a well-characterized CMS system is found in maize. By screening an mtDNA library from cms-T maize with sterile and fertile mtRNA, Dewey et al. (Dewey R E, Levings III C S, Timothy D H (1986) “Novel recombination in the maize mitochondrial genome produces a unique transcriptional unit in the Texas male-sterile cytoplasm.” Cell 44: 439-449) identified a region specific to T-cytoplasm. Said region contains an unusual gene, designated as T-urf13, which is predicted to encode a 13-kDa polypeptide (URF13). T-urf13 is positioned upstream of orf25 and is co-transcribed.
Another example is found in the genus Petunia. The S-pcf gene has been detected in correlation with CMS in Petunias. This locus consists of the 5′ portion of atp9 gene; the exon part of coxII; and an unknown open reading frame, urf-s (Young E G, Hanson M R (1987) “A fused mitochondrial gene associated with cytoplasmic male sterility is developmentally regulated.” Cell 50: 4149).
The specific genes correlated with CMS have also been reported in beans (Johns C, Lu M, Lyznik A, Mackenzie S (1992) “A mitochondrial DNA sequence is associated with abnormal pollen development in cytoplasmic male sterile bean plants.” The Plant Cell 4: 435-449), Brassica (Grelon M, Budar F, Bonhomme S, Pelletier G (1994) “Ogura cytoplasmic male-sterility (CMS)-associated orf138 is translated into a mitochondrial membrane polypeptide in male-sterile Brassica cybrids.” Mol Gen Genet 243: 540-547), radishes (Makaroff C A, Apel I J, Palmer J D (1990) “Characterization of radish mitochondrial atpA-associated sequences and relationship with male sterility.” Plant Mol Biol 15: 735-746), sunflowers (Moneger R, Smart C J, Leaver C J (1994) “Nuclear restoration of cytoplasmic male sterility in sunflower is associated with the tissue-specific regulation of a novel mitochondrial gene.” The EMBO J. 13(1): 8-17), rice (Akagi H (1995) “Genetic diagnosis of cytoplasmic male sterile cybrid plants of rice.” Theor. Appl. Genet. 90:948-951), carrots (Kanzaki H, Takeda M, Kameya T (1991) “Sequence analysis of a mitochondrial DNA fragment isolated from cultured cells of carrot cytoplasmic male-sterile strain.” Japanese J Genet 66: 719-724), and sorghum (Tang H V (1996) “Transcript processing internal to a mitochondrial open reading frame is correlated with fertility restoration in male-sterile Sorghum.” Plant J. 10:123-133).
Although these CMS-associated genes are commonly generated by intra-rearrangement of mtDNA (Hanson M R (1991) “Plant mitochondrial mutations and male sterility.” Annu Rev Genet 25:461-486), the open reading frames share no significant sequence homology. How these genes may act in CMS plants and result in mitochondrial dysfunction and non-functional pollens has not been known until now.
CMS traits are commercially very useful and important in hybrid F1, seed production. This is why transgenic male sterile plants have been attempted and developed by several research groups. For example, Mariani et al. (Mariani C, Beuckeleer J, Trueftner J, Leemans J, Goldberg R B (1990) “Induction of male sterility in plants by a chimeric ribonuclease gene.” Nature 347:737-741) have developed male sterile tobacco by using a tapetum-specific promoter and barnase gene, which functions as a ribonuclease gene in plants. Several experiments have been also attempted to transform these CMS-associated genes into fertile plants. The orf239, CMS-associated mitochondrial DNA sequence in the common bean (Abad A R, Mehrtens B J, Mackenzie S A (1995) “Specific expression in reproductive tissues and fate of a mitochondrial sterility-associated protein in cytoplasmic male sterile beans.” Plant Cell 7:271-285) was used to transform tobacco with or without a mitochondrial targeting sequence. Transformed tobacco exhibited a semi-sterile or male-sterile phenotype even though targeting of the protein to mitochondria has not been made (He S, Abad A R, Gelvin S B, Mackenzie S A (1996) “A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco.” Proc. Natl. Acad. Sci. USA 93:11763-11768). Another CMS-associated gene, urf-s sequence of the pcf gene which encodes the 25 kDa protein in the genus Petunia, had been transformed to petunia and tobacco plants with constructs of mitochondrial targeting sequences. Even though expression of PCF protein was detected in mitochondria of transgenic petunia and tobacco plants, the fertility of the plants was not affected (Wintz H, Chen H C, Sutton C A, Conley C A, Cobb A, Ruth D, Hanson M R (1995) “Expression of the CMS-associated urf-s sequence in transgenic petunia and tobacco.” Plant Mol Biol 28:83-92).
In addition, correct identification of male fertile (N-) cytoplasm and male sterile (S-) cytoplasm in plants including food crops is very important in breeding systems. The estimation of hybrid seed purity and cytoplasmic genotype is conventionally done by the grow-out test (GOT), which is based on the assessment of morphological and floral characteristics (that distinguish the hybrid) in a representative sample of plants that are grown to maturity. For example, pepper plants take several months to reach maturity, and the seeds have to be stored under appropriate conditions as they cannot be marketed until these results become available. In addition, substantial delays can result in the first growing season after hybrid seed production which is taken up by the GOT, which is also the preferred season for hybrid cultivation. In such cases, the seeds have to be stored for up to a year, i.e., until the subsequent growing season, before they can be marketed. For seed companies, large amounts of capital are therefore locked in the form of hybrid seed stocks for prolonged periods while awaiting the results of the GOT. Another disadvantage of the GOT is that it can be subjective due to environmental influences on the expression of morphological characteristics. Further, there is also the possibility that adverse climatic conditions (such as heavy wind or rain, high temperatures, drought) can damage or destroy the crop and make it difficult to collect the data. In order to solve the above problems, a technique using the CMS-associated sequence as a DNA marker has been developed to easily detect a male sterile cytoplasmic type. This technique uses a DNA marker that is detected by Polymerase Chain Reaction (PCR), and it is ideally suited for this purpose as it is much more efficient for handling large numbers of samples than hybridization-based methods like Restriction Fragment Length Polymorphisms (RFLPs).